Full-length huntingtin purification and cleavage of the N-terminal tag

Last week, I completed a purification of the full-length huntingtin protein Q23 from baculovirus expression system (BVES). This protein I hope to use in a series of binding assays to see if I can find any DNA or protein partners of the huntingtin protein. The current construct I have for protein expression (kindly provided by Ihn Sik Seong) has a large N-terminal tag, which is necessary for purification but could interfere with huntingtin binding partner characteristics.

Therefore I made the protein sample both with the tag, as well as with the tag cut off so I can test to see if this makes a difference. The final yields of protein from these parallel purifications were 1 and 0.5 mg of protein respectively. The sample is not as pure as I have previously purified (~90% pure), I believe the Grafix step I usually include helps to clean up the sample considerably. Nonetheless it is sufficiently pure to conduct some preliminary biophysical assays.

Huntingtin samples with the N-terminal tag cut and uncut on 4-12% Bis-Tris SDS-PAGE

You can read all about my work in the lab on Zenodo. Feedback and criticisim, is welcome as always!

 

 

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